The present invention relates generally to the manipulation of genetic materials and, more specifically, to recombinant procedures making possible the production of murine cell lines/strains or transgenic mice possessing desired characteristics.
CD4, expressed predominantly on the surface of human T helper cells, associates with the T cell receptor during T cell activation. In addition, CD4 binds to the gp120 envelope glycoprotein of HIV-1 which results in viral infection of CD4.sup.+ cells. Murine L3T4, the counterpart of human CD4, shares 55% amino acid sequence homology with CD4 in their extracellular domains. However, L3T4 does not bind to gp120, rendering murine T helper cells resistant to HIV-1 infection. Interestingly, human CD4 transfected into murine cells binds HIV-1 on CD4.sup.+ murine cells, but does not facilitate viral infection. The resistance to infection of CD4.sup.+ murine cells is due to a defect in viral entry into the cell. The lack of affinity of gp120 for L3T4 and the inability of human CD4 to permit HIV-1 entry into CD4-transfected murine cells have thwarted efforts to develop a useful transgenic mouse model for HIV-1 infection.
The binding site of gp120 to CD4 has been mapped by site-directed mutagenesis of CD4 and analysis of mutant CD4 interactions with gp120. See, e.g., Clayton et al., Nature 335:363 1988!. The crystal structure of CD4 confirmed that the critical region where CD4 attaches to gp120 is localized to an immunoglobulin-like domain in the C04 extracellular region. Residues 35-83 in domain I of CD4 are the primary contact location of gp120 with CD4. Some believe that amino acids in domain II of CD4 may also be important contact points for gp120 binding to CD4.
The critical region, however, for qp120 binding to CD4 resides between amino acids 43-55. The numbering for the CD4 and L3T4 amino acid sequences in this application is according to that of FIG. 3 in Littman et al., Nature 325:453 1987!. (The numbering is based on L3T4 sequence in that reference.) Mutations introduced in this 13 amino acid span in human CD4 abrogate gp120 binding to the mutated CD4 receptors. Residues 44, 49, 51, 52, 57 and 64 are crucial for efficient gp120 binding to CD4. Residue 87 in CD4 is important for syncytium formation but does not play a role in gp120 binding, nor does it affect viral internalization. Mouse L3T4 and human CD4 are homologous at codons 58, 59, 64, 67 and 87.